Shp2 is involved in neuronal differentiation of hippocampal precursor cells

HiB5 is a multipotent hippocampal stem cell line whose differentiation into cells of a neuronal phenotype is promoted by neurotrophic factors such as PDGF and brain-derived neurotrophic factor (BDNF). We examined the potential role of Src homology 2 (SH2)-containing protein tyrosine phosphatase (Shp2) in this differentiation process. We found that Shp2 became tyrosine phosphorylated following PDGF treatment. Wild-type Shp2 enhanced the expression of neurofilament, synapsin I and PSD95 by PDGF and BDNF, whereas their expression was attenuated by the catalytically inactive mutants Shp2C/S and Shp2DeltaP. Formation of dendritic spine-like structures increased with wild-type Shp2, but diminished with Shp2C/S and Shp2DeltaP. PSD95, localized in the post-synaptic density region of dendritic spines, PDGFRbeta and TrkB were co-immunoprecipitated with Shp2 antibodies. These results suggest that Shp2 plays a positive role in mediating PDGF- and BDNF-activated signaling which promotes the formation of dendritic spines.     

Enhancement by 5-hydroxytryptamine and analogues of desensitization of neuronal and muscle nicotinic receptors expressed in Xenopus oocytes.

1. The action of 5-hydroxytryptamine (5-HT) on neuronal and muscle nicotinic acetylcholine receptors (nicotinic AChR) expressed in Xenopus oocytes was studied. 2. 5-HT enhanced the rate of desensitization of the acetylcholine (ACh) current response in all receptor subtypes investigated (muscle, alpha beta 2 gamma delta and alpha 4 beta 2), acting in a dose-dependent manner. 3. 5-HT also reduced the peak current elicited by ACh in a dose-dependent manner. The IC50 value for the muscle type receptor was 227 +/- 0.44 microM, and 166 +/- 0.47 microM and 283 +/- 0.28 microM for the combinations alpha beta 2 gamma delta and alpha 4 beta 2 respectively. 4. The effect of 5-HT on the responses to ACh (10 microM) was found to be independent of membrane voltage over the range tested (-80 to -10 mV), and to be readily reversed by washout. 5. The action of 5-HT could be mimicked by structurally similar molecules. The homologue tryptamine was less potent than 5-HT in blocking the ACh current, with an IC50 of 1.0 +/- 0.02 mM. Ketanserin, a 5-HT2 receptor antagonist, was more potent than 5-HT, the IC50 being 49.0 +/- 1.4 microM. 6. We postulate that a highly conserved portion of the tertiary structure of nicotinic AChRs, which includes some part of the ACh binding site, has affinity for 5-HT and structural analogues.     

A 62 kDa protein is photoaffinity labelled by [3H]felodipine in vascular smooth muscle, but not in cardiac and skeletal muscle

Irradiation of a cytosolic fraction from vascular smooth muscle in the presence of [3H]felodipine resulted in the labelling of a protein with an apparent molecular weight of 62 kDa. The labelling was seen on UV-irradiation at 360 nm, but not at 254, 278 or at wavelengths above 410 nm. The photolabelling was enhanced in the absence of oxygen. In cytosolic fractions prepared from porcine liver, cardiac and skeletal muscle no photoaffinity labelling of proteins between 90 and 45 kDa could be demonstrated. The results suggest that felodipine is a photoaffinity ligand and that felodipine binds to a soluble protein present in vascular smooth muscle but not in the other tissues tested.     

Involvement of protein phosphatase 2A in arsenic trioxide-induced differentiation and apoptosis of NB4 cells

Arsenic trioxide (ATO) is known to have concentration-dependent dual effects on acute promyelocytic leukemia (APL) cells, preferentially inducing apoptosis at relatively high concentrations and promoting partial differentiation at low concentrations. Protein phosphatase 2A (PP2A) has been demonstrated to take part in the differentiation and apoptosis of malignant hematological cells induced by commonly used medicines, such as all-transretinoic acid (ATRA), interferon, arsenic sulfide, etc. However, there are almost no data on the role PP2A plays in ATO-induced APL cell differentiation/apoptosis. In this report, our goal was to show that ATO inhibited the proliferation and induced the apoptosis and differentiation of neuroblastoma NB4 cells. Okadaic acid (OKA), a specific inhibitor of protein phosphatase activity, markedly increased these effects of ATO on cells. To further elucidate the regulation of PP2A during ATO-induced differentiation/apoptosis of NB4 cells, we measured the phosphatase activity and protein expression of PP2A. The activity of PP2A in NB4 cells decreased with increasing concentration of ATO. This decrease of PP2A activity appeared to parallel phenotypic and functional changes of NB4 cells. Western blot analysis showed that the levels of the PP2A structural subunit PP2A-A decreased during the course of ATO-induced differentiation/apoptosis, whereas the expression of the B and C subunits of PP2A was relatively unaltered. In conclusion, the decrease of PP2A activity may be involved in ATO-induced apoptosis and differentiation of APL cells, and this decrease is predicted to be related to the repression of PP2A-A subunit expression.     

Steroid inhibition of rat neuronal nicotinic alpha4beta2 receptors expressed in HEK 293 cells

Steroids, in addition to regulating gene expression, directly affect a variety of ion channels. We examined the action of steroids on human embryonic kidney 293 cells stably transfected to express rat alpha4beta2 neuronal nicotinic receptors. Each steroid that was tested inhibited acetylcholine responses from these receptors, with slow kinetics requiring seconds for block to develop and recover. The action of one steroid [3alpha,5alpha, 17beta-3-hydroxyandrostane-17-carbonitrile (ACN)] was studied in detail. Block showed enantioselectivity, with an IC(50) value of 1.5 microM for ACN and 4.5 microM for the enantiomer. Inhibition curves had Hill slopes larger than 1, indicating more than one binding site per receptor. Block did not require intracellular compounds containing high-energy phosphate bonds and was not affected by analogs of GTP, suggesting that the mechanism does not require the activation of second messengers. Block did not appear to be strongly selective between open and closed channel states or to involve changes in desensitization. A comparison of different steroids showed that a beta-orientation of groups at the 17 position produced more block than alpha-orientated diastereomers. The stereochemistry at the 3 and 5 positions was less influential for block of alpha4beta2 nicotinic receptors, despite its importance for potentiation of gamma-aminobutyric acid(A) receptors. The ability of steroids to block neuronal nicotinic receptors correlated with their ability to produce anesthesia in Xenopus tadpoles, but the concentrations required for inhibition are generally greater. Similarly, the concentrations of endogenous neurosteroids required to inhibit receptors are larger than estimates of brain concentrations.     

锌指蛋白A20对大鼠急性重症胰腺炎LPS-TLR4信号通路的影响

目的 研究锌指蛋白A20对大鼠急性重症胰腺炎LPS-TLR4信号通路的影响.方法 采用5％牛磺胆酸钠溶液胰胆管注射诱发急性重症胰腺炎（SAP）大鼠模型,24只大鼠按数字表法随机分为三组.A组（假手术组）、B组（SAP模型组）、C组（SAP＋ LPS组）,每组8只.免疫组织化学法检测胰腺组织TLR4、NF-κBp65和p38MAPK的水平.结果 与A组相比,B、C组胰腺组织中锌指蛋白A20表达明显下降（t=17.234、19.698,均P＜0.05）,而TLR4、NF-κBp65和p38MAPK表达均明显增加（t=15.909、20.432、16.543、18.629、22.105、19.006,均P＜0.05）.C组与B组相比锌指蛋白A20表达明显下降（t=14.894,P＜0.05）,而TLR4、NF-κBp65和p38MAPK表达均明显增加（t=14.047、15.582、17.070,均P＜0.05）.结论 SAP时胰腺锌指蛋白A20的表达减少,对LPS-TLR4通路的抑制作用减弱,使胰腺损伤加重.     

A novel SGLT is expressed in the human kidney

Selective inhibitors of sodium-glucose cotransporter 2 (SGLT2)-mediated reabsorption of glucose in the proximal tubule of the kidney are being developed for the treatment of diabetes. SGLT2 shares high degree of homology with SGLT3; however, very little is known about the expression and functional role of SGLT3 in the human kidney. Indeed, the SGLT2 inhibitors that are currently in clinical trials might affect the expression and/or the activity of SGLT3. Therefore, the present study examined the expression of SGLT3 mRNA and protein in human kidney and in a human proximal tubule HK-2 cell line. The results indicated that human SGLT3 (hSGLT3) message and protein are expressed both in vivo and in vitro. We also studied the activity of hSGLT3 protein following its over-expression in mammalian kidney-derived COS-7 cells and in HK-2 cells treated with the imino sugar deoxynojirimycin (DNJ), a potent agonist of hSGLT3. Over-expression of hSGLT3 in COS-7 cells increased intracellular sodium concentration by 3-fold without affecting glucose transport. Activation of hSGLT3 with DNJ (50μM) increased sodium uptake in HK-2 cells by 5.5 fold and this effect could be completely blocked with SGLT inhibitor phlorizin (50μM). These results suggest that SGLT3 is expressed in human proximal tubular cells where it serves as a novel sodium transporter. Up-regulation of the expression of SGLT3 in the proximal tubule in diabetic patients may contribute to the elevated sodium transport in this segment of the nephron that has been postulated to promote hyperfiltration and renal injury.     

SDS3 interacts with ARNT in an AhR ligand-specific manner regulating expression of cKrox and S100A4 in CD4+CD8+ DPK thymocytes differentiation

To study mechanisms underlying differential effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (B(a)P) on thymocyte differentiation, we examined effects of AhR ligands on the differentiation of DPK cells, a CD4⁺CD8⁺ thymic lymphoma cell line which can differentiate into CD4⁺CD8⁻ thymocytes. In contrast to TCDD, which inhibited the differentiation, B(a)P showed little effect. Antigen-mediated up-regulation of S100A4, S100A6, galectin-1, and TRAF5-like protein was remarkably suppressed by TCDD, but slightly by B(a)P. Immunoprecipitation using anti-ARNT Ab revealed that SDS3, a component of the Sin3/HDAC repressor complex, was associated with ARNT only when DPK cells were incubated with TCDD. Expression of cKrox S100A4 was derepressed when SDS3 protein was reduced. These results indicate that although it is generally known that many AhR ligands such as TCDD and B(a)P function mainly by the AhR/ARNT complex, ligand-specific interaction between SDS3 and ARNT exerts differential effects on the expression of genes associated with thymocyte differentiation.     

Calcineurin homologous protein isoform 2 (CHP2), Na+/H+ exchangers-binding protein,is expressed in intestinal epithelium

The Na+/H+ exchangers (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of Na+ and H+. Calcineurin homologous protein (CHP) acts as a crucial cofactor for NHE activity through direct interaction with the carboxyl-terminal tail region of NHEs. We have cloned a new rat CHP isoform (rCHP2) and characterized the binding property to NHEs and the tissue distribution. rCHP2 binds to the juxtamembrane region of plasma membrane-type NHE isoforms (NHE1-5) in vivo and in vitro as well as rCHP1 (original rat CHP). Interestingly, CHP2 is predominantly expressed in the small and large intestine although rCHP1 shows relatively ubiquitous expression at both the mRNA and protein levels. In situ hybridization experiments demonstrated the abundant expression of CHP2 in the epithelial cell layer of villi of the small intestine in contrast with the expression of CHP1 in both the epithelial layer and connective tissues. These results suggest that CHP2 functions in the absorptive epithelium for the intestine with NHE(s).     

锌指蛋白A20在过敏性哮喘中的研究进展

锌指蛋白A20(也称TNFAIP3)是一种可在多种炎症条件,如LPS、IL-1、TNF-α等刺激下,被激活的具有去泛素化和泛素化双重功能的胞质蛋白。目前,A20被认为是NF-κB的负调控因子,具有免疫调节作用。最新研究表明,A20参与了过敏性哮喘的发生和发展。因此,该文对A20的起源、结构、生物学功能及其在过敏性哮喘中的作用进行综述,以期为过敏性哮喘的治疗和预防提供新的方法和思路。     

Karanjin from Pongamia pinnata induces GLUT4 translocation in skeletal muscle cells in a phosphatidylinositol-3-kinase-independent manner

Insulin-stimulated glucose uptake in skeletal muscle is decreased in type 2 diabetes due to impaired translocation of insulin-sensitive glucose transporter 4 (GLUT4) from intracellular pool to plasma membrane. Augmenting glucose uptake into this tissue may help in management of type 2 diabetes. Here, the effects of an identified antihyperglycemic molecule, karanjin, isolated from the fruits of Pongamia pinnata were investigated on glucose uptake and GLUT4 translocation in skeletal muscle cells. Treatment of L6-GLUT4myc myotubes with karanjin caused a substantial increase in the glucose uptake and GLUT4 translocation to the cell surface, in a concentration-dependent fashion, without changing the total amount of GLUT4 protein and GLUT4 mRNA. This effect was associated with increased activity of AMP-activated protein kinase (AMPK). Cycloheximide treatment inhibited the effect of karanjin on GLUT4 translocation suggesting the requirement of de novo synthesis of protein. Karanjin-induced GLUT4 translocation was further enhanced with insulin and the effect is completely protected in the presence of wortmannin. Moreover, karanjin did not affect the phosphorylation of AKT (Ser-473) and did not alter the expression of the key molecules of insulin signaling cascade. We conclude that karanjin-induced increase in glucose uptake in L6 myotubes is the result of an increased translocation of GLUT4 to plasma membrane associated with activation of AMPK pathway, in a PI-3-K/AKT-independent manner.     

Indocarbazostatins C and D, new inhibitors of NGF-induced neuronal differentiation in PC12 cells

Indocarbazostatins C (3) and D (4), new inhibitors of NGF-induced neurite outgrowth were isolated from culture broth of a mutant strain, Streptomyces sp. MUV-6-83. The structural elucidation of 3 and 4 revealed that these inhibitors were methyl ester analogs of the corresponding ethyl ester compounds, indocarbazostatin (1) and indocarbazostatin B (2), respectively.     

Expression of CYP3A4 mRNA is correlated with CYP3A4 protein level and metabolic activity in human liver

A relatively large amount of human liver tissue was required to determine the exact activity of human hepatic CYP3A. Although, the quantity of available human liver tissue samples is limited. We measured levels of CYP3A4 mRNA by RT-PCR with a radiolabeled primer specific for CYP3A4 and compared mRNA expression with CYP3A4 protein level and metabolic activity in liver. The level of CYP3A4 mRNA was correlated with the levels of CYP3A4 protein and activity. Our results suggest that CYP3A4 protein and activity levels can be predicted from CYP3A4 mRNA levels determined by RT-PCR and using a very small amount of liver tissue.     

The histamine H4 receptor is functionally expressed on neurons in the mammalian CNS

Background and purpose:

The histamine H₄ receptor is the most recently identified of the G protein-coupled histamine receptor family and binds several neuroactive drugs, including amitriptyline and clozapine. So far, H₄ receptors have been found only on haematopoietic cells, highlighting its importance in inflammatory conditions. Here we investigated the possibility that H₄ receptors may be expressed in both the human and mouse CNS.

Methods:

Immunological and pharmacological studies were performed using a novel anti-H₄ receptor antibody in both human and mouse brains, and electrophysiological techniques in the mouse brain respectively. Pharmacological tools, selective for the H₄ receptor and patch clamp electrophysiology, were utilized to confirm functional properties of the H₄ receptor in layer IV of the mouse somatosensory cortex.

Results:

Histamine H₄ receptors were prominently expressed in distinct deep laminae, particularly layer VI, in the human cortex, and mouse thalamus, hippocampal CA4 stratum lucidum and layer IV of the cerebral cortex. In layer IV of the mouse somatosensory cortex, the H₄ receptor agonist 4-methyl histamine (20 µmol·L⁻¹) directly hyperpolarized neurons, an effect that was blocked by the selective H₄ receptor antagonist JNJ 10191584, and promoted outwardly rectifying currents in these cells. Monosynaptic thalamocortical CNQX-sensitive excitatory postsynaptic potentials were not altered by 4-methyl histamine (20 µmol·L⁻¹) suggesting that H₄ receptors did not act as hetero-receptors on thalamocortical glutamatergic terminals.

Conclusions and implications:

This is the first demonstration that histamine H₄ receptors are functionally expressed on neurons, which has major implications for the therapeutic potential of these receptors in neurology and psychiatry.     

Low 4-aminopyridine concentration-induced contraction is mediated by neuronal noradrenaline in canine saphenous vein

4-Aminopyridine (4-AP), a known inhibitor of the voltage-dependent potassium channels, is able to increase the basal tone of different types of blood vessel preparations. In order to determine the efficiency of 4-AP in veins and to clarify its possible mechanism of action, the aim of the present study was to determine the basal tone and release of radio-labelled tissue noradrenaline (NA) after administration of low 4-AP concentrations. Experiments were performed in canine saphenous vein in the absence and presence of functional endothelium. 4-AP (0.012-5 microM) enhanced the basal tone of venous rings without and with endothelium (maximum tone at 5 microM 4-AP: 2.20 +/- 1.29 and 1.3 +/- 0.57 mN, respectively). NA stores of the venous tissue were loaded by adding 1 mM NA to the tissue for 10 min and then washed out. After loading the NA-stores of venous tissue, 4-AP-induced contractions were significantly increased both in the absence and presence of endothelium (maximum tone at 5 microM 4-AP after loading with NA: 10.51 +/- 3.64 and 10.52 +/- 4.69 mN, respectively). Following NA loading, chemical denervation of the endothelium denuded venous preparations by 0.5 mM 6-hydroxydopamine (6-OHDA) completely abolished the contractions evoked by 4-AP. After incubation of the saphenous preparations with 3H-NA, 5 microM 4-AP significantly increased tritium-efflux from the tissue. These results provide evidence for the efficiency of 4-AP on the basal tone of isolated canine saphenous vein when applied in low concentrations. Furthermore, it is suggested that this action of 4-AP may considerably depend on the release of NA from the perivascular nerve endings.     

MDMA impairs mitochondrial neuronal trafficking in a Tau- and Mitofusin2/Drp1-dependent manner

Identification of the mechanisms by which drugs of abuse cause neuronal dysfunction is essential for understanding the biological bases of their acute and long-lasting effects in the brain. Here, we performed real-time functional experiments of axonal transport of mitochondria to explore the role of in situ mitochondrial dysfunction in 3,4-methylenedioxymethamphetamine (MDMA; “ecstasy”)-related brain actions. We showed that MDMA dramatically reduced mitochondrial trafficking in hippocampal neurons in a Tau-dependent manner, in which glycogen synthase kinase 3β activity was implicated. Furthermore, we found that these trafficking abnormalities were rescued by over-expression of Mitofusin2 and dynamin-related protein 1, but not of Miro1. Given the relevance of mitochondrial targeting for neuronal function and neurotransmission, our data underscore a novel mechanism of action of MDMA that may contribute to our understanding of how this drug of abuse alters neuronal functioning.